This invention relates to polypeptides which, like antibiotics, can be employed to inhibit the spread of bacterial infections in mammals caused by gram-positive bacteria.
One aspect of the structure of gram-positive bacteria including, for example, bacteria such as streptococci and staphylococci which are responsible for such mammalian infections as strep throat, rheumatic fever, nephritis, toxic shock syndrome and endocarditis is the presence of virulence determinants responsible for infecting the host cell. These determinants are essential factors for the spread of infection following any of a number of mechanisms known to those skilled in the art. They may take the form of enzymes, anti-phagocytotic agents, adhesive molecules and others. One form of virulence determinant is the M-protein which is a protein attached to the surface of gram-positive streptococci. The streptococcal M-protein is a coiled-coil fibrillar structure extending about 60 nm from the streptococcal cell wall.
This invention arises from structural studies of the M protein and other virulence determinants located on the surface of gram positive bacteria and the mechanisms by which such surface proteins are anchored to the cell.
FIG. 2 is a schematic representation of the coiled-coil M protein molecule on the streptococcal cell wall. The molecule which comprises approximately 441 amino acid residues is shown with a variable amino end and a conserved carboxyl end, the so called N-terminal and C-terminal. The protein is shown as extending from the outer surface of the cell. The surface of the cell comprises the cell membrane and the cell wall which is composed of peptidoglycan and group carbohydrate. The N-terminal end of the molecule is more exposed to the immune system, and the variability of the N-terminal region is the principal reason why there is difficulty in preparing a vaccine effective against all strains of streptococci.
This invention is principally concerned with that region of the C-terminal of the M-protein which, in FIG. 2, is shown at the interface between the cell wall and the cell membrane and penetrating into the cell membrane.
Surface proteins in both gram-positive and gram-negative bacteria are generated in the cytoplasm, transferred through the membrane and anchored at their C-terminal ends to the membrane. If the anchor is missing the protein will escape into the growth media with gram-positive bacteria. In gram-negative bacteria, the protein becomes trapped in the periplasmic space between the cell wall and the outer membrane.
To understand the basis for this invention, it is necessary to consider in some detail the structure of the C-terminal of the M-protein and to understand from these considerations the nature of the anchoring mechanism of the M-protein to the cell surface.
FIG. 1A shows in the first line of the top section the amino acid sequence of the M6-protein from position 375 to 441 at the C-terminal. It also shows in subsequent lines the amino acid sequences at the C-terminal of several other gram-positive bacteria, the significance of which will become apparent as the discussion progresses.
The lower portion of FIG. 1B shows the oligonucleotide structure of selected portions of each of the genes which express the polypeptides shown in the upper section. In the figure, the standard single letter codes are employed to identify the amino acids and the bases.
Attention is directed to the shaded areas of the upper section. It will be noted that in most of the polypeptides shown, the sequence LPSTGE is highly conserved. This observation is most important to the understanding of this invention.
Reading from the leucyl residue (L) at the N-terminal end of the shaded area of FIG. 1A, the C-terminal of the M6 protein is composed of the LPSTGE region; a linker region, TAN; a hydrophobic region of twenty amino acids, PFFTAAALTVMATAGVAAVV; followed by a highly charged tail region, KRKEEN.
It will be noted that in each of the polypeptides shown, which are, in fact, segments of known surface molecules some of which are virulence determinants on the surface of gram-positive bacteria, there is a most highly conserved region, principally LPSTGE, a relatively highly conserved hydrophobic region containing from about 15 to 20 amino acid residues, a charged tail region containing from about 4 to 6 amino acid residues and a linker region joining the LPSTGE region to the hydrophobic region. This linker region contains from about 3 to 7 amino acids.
This homology, of course, reflects comparable conserved regions in the oligonucleotides as shown in the lower part of FIG. 1B.
To study these structures and their importance in designing polypeptides useful to inhibit the spread of infection caused by gram-positive bacteria it was necessary to produce a number of products including genes, plasmids and E. coli vectors carrying the plasmids.
Table I lists several of these plasmids and the characteristic features of the proteins expressed by E. coli carrying each plasmid.
The nature of the proteins was determined by several factors including the genetic structure of the plasmid used to express them, gel electrophoresis using selected markers and antibodies. The details of these studies are given in the experimental section.
Three general types of plasmids were produced. One set based on M6.1, one set based on PIII or PIII fused to M6.1, and a final set based on PhoA or PhoA fused with M6.1. The three basic plasmids and methods for their production are all known. The proteins produced by the expression of the various plasmids are generally characterized by the presence or absence of the LPSTGE region and/or the hydrophobic region.
The various plasmids were expressed from selected strains of E. coli which were then analyzed to determine the position of the expressed protein on the outer cell surface or in the periplasm. The fact that the protein is on the outer surface is established by the experiment described at pages 21, lines 1 through 12. The results of the analysis appear beneath the table.
TABLE 1 ______________________________________ Plasmid Protein ______________________________________ pM6.1 M6.sup.1 pM6.1.sub.1-406 M6 minus LPSTGE, hydrophobic region and charged tail.sup.2 pM6.1.sub..DELTA.LPSTG M6 minus LPSTGE, but including the hydrophobic region and the charged tail.sup.3 pNDI PIII.sup.4 pND372 PIII gene devoid of its own membrane anchor.sup.5 pPIII:M6.1.sub.367-441 Contains LPSTGE, hyrdrophobic region and charged tail of M6 fused to position 375 of pIII.sup.6 pPIII:M6.1.sub.413-441 Contains hydrophobic and tail region of M6 fused to position 375 of PIII, but no LPSTGE region.sup.7 pPhoA pPhoA protein, with no anchor.sup.8 pPhoA:M6.1.sub.303-441 Contains LPSTGE, hydrophobic and tail regions of M6 fused to C-terminal of pHoA.sup.9 pPhoA:M6.1.sub.414-441 Contains hydrophobic and tail regions of M6, but no LPSTGE, fused to C-terminal of phOA.sup.10 ______________________________________
Proteins expressed by these plasmids:
1. Found in membrane fraction. PA0 2. Secreted in periplasm, but not found on membrane. PA0 3. Secreted in periplasm, but not found on membrane. PA0 4. Found in membrane fraction. PA0 5. Secreted in periplasm, but not found on membrane. PA0 6. Found in membrane fraction. PA0 7. Secreted in periplasm, but not found on membrane. PA0 8. Secreted in periplasm, but not found on membrane. PA0 9. Found in membrane fraction. PA0 10. Found in periplasm, but not on membrane.
From a study of these results, it can be concluded that the M protein will attach itself to the cell membrane when both the LPSTGE region and the hydrophobic region are present. The hydrophobic region alone is not sufficient for anchoring. The LPSTGE region, or an analog is responsible for anchoring the Mprotein. This invention is based on that discovery.